Fig. 9

Loss of endothelial Notch1 augments atherosclerotic plaque burden in a model of hypercholesterolemia. a Timeline for mouse model of hypercholesterolemia. b Quantification of liver LDLR protein levels by immunoblot to confirm PCSK9 activity in PCSK9-AAV-injected animals compared to control-AAV-injected animals at time of harvest (PDI used as loading control). Each lane corresponds to one animal. c Circulating cholesterol levels at time of evaluation (12 weeks after AAV injection) (n = 6–11 per group, each dot corresponding to one animal). Indicated are individual cholesterol levels in each group. d Representative aortas from each of the four groups: Control + AAV CT, Notch1 ^ECKO + AAV CT, Control + PCSK9, and Notch1 ^ECKO + PCSK9. Control animals were Cre⁺ tdTomato reporter. e Example of aorta stained with Sudan IV for quantification of atherosclerotic plaque area and identification of the arch and descending aorta regions. f Quantification of plaque burden in the whole aorta, arch, and descending aorta was determined by calculating the percent of aortic surface area covered by atherosclerotic lesions in each group (n = 6 for Control and n = 9 for Notch1 ^ECKO). Each point represents the lesion area per mouse. The mean area for each group of mice is indicated by the horizontal bars. T-test *P < 0.05, **P < 0.01, ns = not significant. g En face imaging of wild-type (C57BL/6) adult mouse aorta reveals enhanced nuclear presence of Notch1 (red) in endothelial cells of the descending aorta compared to endothelial cells of the lower arch (representative images of n = 6). Scale bar = 20 µm. h Venus Notch reporter mouse aorta imaged en face to assess Notch signaling comparing the descending aorta to the arch (representative images of n = 3). Scale bar = 20 µm. i Protein lysates from bovine aorta (descending aorta, lower arch and smooth muscle) were analyzed by immunoblot for expression of Notch1 intracellular domain (NICD) and Klf2 using Ve-Cadherin as loading control. Each lane shows isolation from a distinct biological replicate. NICD levels trend with Klf2 expression level differences between the descending aorta and lower arch (data from nine technical replicates isolated from four biological replicates are shown on graph, T-test ****P < 0.0001)